Novel methods for modulating inflammatory and/or immune responses

ABSTRACT

The invention is directed to novel methods for modulating inflammatory and/or immune responses. Such methods utilize compositions comprising extraembryonic cells (herein referred to as EE cells) including but not limited to extraembryonic HLA-G positive cells (herein referred to as EHP cells) and amnion-derived multipotent progenitor cells (herein referred to as AMP cells); compositions comprising expanded EE cell populations, and/or cell lysates and/or conditioned media derived therefrom, alone or in combination with each other and/or in combination with various extracellular matrices and/or devices and/or other suitable active agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 USC §119(e) to U.S.Provisional Application No. 60/880,745, filed Jan. 17, 2007, U.S.Provisional Application No. 60/902,440, filed Feb. 21, 2007, U.S.Provisional Application No. 60/997,604, filed Oct. 4, 2007, the contentsof which are incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in part with United States government supportawarded by the following agency: U.S. Army Medical Research AcquisitionActivity, ERMS #06100002. The United States may have certain rights tothis invention.

FIELD OF THE INVENTION

The field of the invention is directed to novel methods for modulatinginflammatory and/or immune responses. Such methods utilize compositionscomprising extraembryonic cells (herein referred to as EE cells)including but not limited to extraembryonic HLA-G positive cells (hereinreferred to as EHP cells) and amnion-derived multipotent progenitorcells (herein referred to as AMP cells); compositions comprisingexpanded EE cell populations, and/or cell lysates and/or conditionedmedia derived therefrom, alone or in combination with each other and/orin combination with various extracellular matrices and/or devices and/orother suitable active agents.

DESCRIPTION OF RELATED ART

U.S. Published Application No. 2006026337 (incorporated herein byreference) discloses the immunomodulatory properties of multipotentadult progenitor cells, called MAPCs, and uses thereof.

Ueta, M., et al., (Clin Exp Immunol 2002; 129:464-470) describe theimmunosuppressive properties of decellularized amniotic membrane.

Klyushnenkova, E., et al., (Journal of Biomedical Science, 2005,12:47-57) describe T cell responses to allogeneic human mesenchymal stemcells, called MSCs.

Williams, M. (Journal of Hematotherapy & Stem Cell Research, 2003,12:757-758) discusses the functional expression of HLA-G and whether itcan be exploited for successful stem cell transplantation andengraftment.

Götherström, C., et al., (The Hematology Journal, 2005, 90(8):1017-1026)disclose that adult bone marrow-derived mesenchymal stem cells do notexpress HLA-G protein.

BACKGROUND OF THE INVENTION

Stem cells have the remarkable potential to proliferate anddifferentiate into many different cell types in the body. Serving as arepair system for the body, they can theoretically divide without limitto replenish other cells throughout a person's life. When a stem celldivides, each new cell has the potential to either remain a stem cell orbecome another type of cell with a more specialized function, such as amuscle cell, a red blood cell, or a brain cell. Perhaps the mostimportant potential application of human stem cells is the generation ofcells and tissues that could be used for cell-based therapies. Examplesof stem cell studies are provided (Tylki-Szymanska, A., et al., Journalof Inherited Metabolic Disease, 1985. 8(3): p. 101-4; Yeager, A. M., etal., American Journal of Medical Genetics, 1985. 22(2): p. 347-55; John,T., 2003. 16(1): p. 43-65, vi.).

Placental tissue is abundantly available as a discarded source of a manypotentially useful cell types including a type of multipotent cellcalled placental-derived cells. Although discarded at parturition aspart of the placental membranes, lineage analysis shows that, theepithelial layer of the amnion, from which such multipotent cells can beisolated, is uniquely descended from the epiblast in embryonicdevelopment. The epiblast contains the cells that will ultimatelydifferentiate into the embryo and cells that will give rise to anextraembryonic tissue, the amnion. Thus far, only four cell types havebeen described in the literature as being pluripotent. These are theinner cell mass (ICM) of the pre-implantation embryo, which gives riseto the epiblast, the epiblast itself, embryonic stem (ES) and embryonicgerm cells (EG). Thus, identification, purification and propagation of amultipotent cell population from discarded amnion tissue would providean extremely valuable source of stem cells for replacement cell therapy.

With an average yield of over 200 million cells per placenta, largenumbers of cells are available from this source. If these cells were tobecome useful cells for transplantation medicine, they could provide anearly inexhaustible supply of starting material in every part of theworld. No stem cell source provides such a large starting population ofcells, and collection does not require an invasive or destructiveprocedure. Furthermore, there are no ethical, religious or social issuesassociated with these cells as the tissue is derived from the placenta.

Another important consideration in stem cell and organ transplanttherapies is graft tolerance. In humans, the protein expression of thecell surface marker HLA-G was originally thought to be restricted toimmune-privileged sites such as placenta, as well as related cells,including some isolated from amniotic fluid, placental macrophages, andcord blood, thus implicating its role in maternal-fetal tolerance(Urosevic, M. and Dummer, R. (2002) ASHI Quarterly; 3rd Quarter2002:106-109). Additionally, studies involving heart-graft acceptancehave suggested that the protein expression of HLA-G may enhance grafttolerance (Lila, N., et al. (2000) Lancet 355:2138; Lila, N. et al.(2002) Circulation 105:1949-1954). HLA-G protein is not expressed on thesurface of undifferentiated or differentiated embryonic stem cells(Drukker, M, et al. (2002) PNAS 99(15):9864-9869). Thus, it is desirablethat stems cells intended for cell-based therapies express HLA-Gprotein.

The transfer of living cells, tissues, or organs from a donor to arecipient, with the intention of maintaining the functional integrity ofthe transplanted material in the recipient defines transplantation. Amajor goal in solid organ transplantation is the permanent engraftmentof the donor organ without a graft rejection immune response generatedby the recipient, while preserving the immunocompetence of the recipientto respond to other foreign antigens. Typically, in order to preventhost rejection responses, nonspecific immunosuppressive agents such ascyclosporine, methotrexate, steroids and FK506 are used. These agentsmust be administered on a daily basis and if stopped, graft rejectionusually results. Despite the use of immunosuppressive agents, chronicgraft rejection still remains a major source of morbidity and mortalityin human organ transplantation. Most human transplants fail within 10years without permanent graft acceptance. Only 50% of heart transplantssurvive 5 years and 20% of kidney transplants survive 10 years (Opelz,et al., Lancet, 1:1223 (1981); Gjertson, UCLA Tissue Typing Laboratory,p. 225 (1992); Powles, Lancet, p. 327 (1980); and Ramsay, New Engl. J.Med., p. 392 (1982)).

Among the most prominent adverse reactions encountered as a result oftransplant therapies are (i) the host versus graft response (“HVG”)(rejection of the transplant by an immune competent host), and (ii)graft versus host disease (“GVHD”) (which occurs primarily in animmunocompromised host when it is recognized as non-self byimmunocompetent cells in the graft). Graft rejection in a host can beavoided by perfectly matching the donor and the host tissue. However,perfect matches are virtually non-existent (with the exception ofidentical twins). One potential way around this is the use of autologous(syngeneic) tissue. Unfortunately, the host tissue is often not suitableor was not collected prior to need. In fact, the need for the transplanttherapy is frequently to replace damaged tissue in the host. This meansthat the use of autologus (syngeneic) tissue is not generally useful inpractical applications.

Another option is matching an allogeneic donor and host as closely aspossible using blood and/or tissue typing. Unfortunately, even theclosest of matches does not prevent serious HVG, so allogeneictransplant therapies require immunosuppression and immunosuppressivedrugs (see below).

Another approach to avoid HVG and its complications in transplanttherapies is to disable the immune system of the recipient host. A drawback to such immunoablation or suppression is that it compromises thehost's immune defenses such that the host is readily susceptible toinfections, a major cause of morbidity and mortality among transplantpatients. Compromising the host immune system also causes or exacerbatesgraft versus host disease (“GVHD”). GVHD occurs when donor tissuecontains immunocompetent cells that recognize MHC proteins of therecipient as non-self. This activates T-cells called TH1 cells which inturn secrete pro-inflammatory cytokines, such as IL-2, interferon gamma,and TNF alpha, which trigger an immune attack on recipient targetsincluding the skin, GI tract, liver, and lymphoid organs (Ferrara andDeeg, 1991). GVHD is particularly a problem in bone marrow transplants,where it has been shown to be mediated primarily by T lymphocytes (Grebeand Streilein, 1976).

A number of immunosuppressive drugs have been developed and are in useto prevent and/or treat these immune system dysfunctions. Unfortunately,none of the immunosuppressive drugs currently available are entirelyeffective and all of them have serious drawbacks and deleterious sideeffects. Glucocorticoids, which are used primarily to treat inflammationand inflammatory diseases, are known to be immunosuppressive and areconsidered be the best primary treatment for HVG and GVHD. They inhibitT-cell proliferation and T-cell-dependent immune responses. Drugs thatact on immunophilins (i.e. cyclosporine, tacrolimus, sirolimus) can beeffective in reducing adverse immune reactions in transplant patients,but they also weaken the immune system so much that patients are lefthighly vulnerable to infections. Cytostatics (i.e. methotrexate,azathiopine, mercatopurine, and cytotoxic antibiotics) are also widelyused either alone or in combination with other drugs. They cause avariety of side effects, some of which can be deleterious to thepatient.

Antibodies (polyclonals and monoclonals such as anti-T-cell receptor(CD23) and anti-IL2 receptor (CD25) antibodies) have also been used.Many other drugs have also been used (i.e. interferon, opioids, TNFbinding proteins, mycophenolate, and small biological agents such asFTY720). None of the immunosuppressive drugs, whether used alone or incombination with other agents, are fully effective and all of themgenerally leave patients still susceptible to HVG and GVHD and weakentheir ability to defend against infection. Furthermore, all of thesedrugs cause serious side effects including gastrointestinal toxicity,nephrotoxicity, hypertension, myelosuppression, hepatotoxicity, andhypertension, to name a few.

Clearly, a more specific type of immune suppression without thedrawbacks listed above would be ideal. For example, an agent that cansuppress or eliminate alloreactive T-cells, specifically, would beeffective against HVG and GVHD (at least for allogeneic grafts) withoutthe negative side effects that occur with agents that generally attackand compromise the immune system. However, to date, no such agent(s)have been developed. Therefore, it is an object of the present inventionto fulfill this unmet need.

SUMMARY OF THE INVENTION

In accordance with the present invention, Applicants have discoveredthat extraembryonic cells (EE cells) including but not limited toextraembryonic HLA-G positive cells (EHP cells) and amnion-derivedmultipotent progenitor cells (AMP cells), and/or cell lysates and/orconditioned media derived therefrom, alone or in combination with eachother and/or other suitable active agents, are useful agents capable ofsuppressing, preventing, ameliorating or treating HVG, GVHD, as well asmany other immune and/or inflammatory diseases and disorders. The cellsof the present invention express HLA-G, do not express MHC Class IIantigens, are telomerase negative, do not form teratomas, are notimmortal, secrete cellular modulatory factors, and are readily availablein great numbers.

It is an object of the instant invention to provide methods formodulating inflammatory and/or immune responses by administering EHPcells, and in particular, AMP cells. It is also an object of the instantinvention to suppress, treat, prevent and/or ameliorate inflammatory,immune, and/or allergic diseases and disorders in a subject in needthereof by administering EHP cells, and in particular, AMP cells. It isa further object of the invention to provide methods for modulatinginflammatory and/or immune responses and/or treating, preventing and/orameliorating inflammatory, immune, and/or allergic diseases anddisorders in a subject in need thereof by administering conditionedmedia derived from EHP cells, cell lysates derived therefrom, or cellproducts derived therefrom, each alone or in combination, including incombination with each other and/or other suitable active agents. It is afurther object of the invention to provide methods for modulatinginflammatory and/or immune responses and/or treating, preventing and/orameliorating inflammatory, immune, and/or allergic diseases anddisorders in a subject in need thereof by administering conditionedmedia derived from AMP cells, referred to herein as amnion-derivedcytokine solution (ACCS), cell lysates derived therefrom, or cellproducts derived therefrom, each alone or in combination, including incombination with each other and/or in combination with variousextracellular matrices and/or devices and/or other suitable activeagents.

The phenotypical characterization of AMP cells reveals them to be idealcandidates for cellular therapy for immune-mediated diseases anddisorders. As shown in the example below, in vitro data show AMP cellsare not immunogenic and have immuno-modulatory properties. AMP cellsdown-regulate T cell responses to various stimuli, including mitogenresponses, allo-antigen (MLR), and memory T cell responses. Themechanisms by which AMP cells may facilitate a down-regulatedimmuno-modulated environment may include several aspects. First, AMPcells up-regulate the expression of the programmed cell death ligandsPD-L1 and PD-L2 when exposed to proinflammatory cytokines such as IFN-γ.These ligands may bind to their receptors (PD-1) on T cells, resultingin the down-regulation of activation and cytokine secretion. AMP cellsare also positive for the expression of Fas antigen. This antigen caninteract with Fas-ligand expressed on activated T cells and instigatecell death of these T cells. Finally, AMP cells have high expression ofHLA-G surface antigen when exposed to IFN-γ and proinflammatorycytokines. In fact, AMP cells up-regulate HLA-G expression on theirsurface during culture in a MRL. HLA-G has been shown to havesubstantial immuno-modulatory functions, including impairment ofproliferation of allo-specific T cells, inhibition of NK cell activity,tolerization of dendritic cells, and induction of T regulatory cells. Asshown in the example below, AMP cells do not have immuno-modulatoryeffects on T cells when separated from these responding cells viatranswell membranes. Thus, the mechanisms of action by AMP cells mostlikely involve cell-to-cell contact with responding immune mediatingcells. These unique characteristics of AMP cells identify them as anideal cellular therapy for afflictions involving immune-mediatedmechanisms.

Accordingly, a first aspect of the invention is a method of suppressing,preventing or ameliorating an immune response in a subject in needthereof, such method comprising administering to the subject aneffective amount of a composition selected from the group consisting ofa composition comprising EHP cells, including AMP cells, conditionedmedia derived therefrom, cell lysates derived therefrom, and cellproducts derived therefrom, each alone or in combination with eachother. In specific embodiments, the immune response to be suppressed,prevented or ameliorated is T cell activation, NK cell activation,downregulation of antigen presenting cell activation, or tolerization ofdendritic cells to prevent T cell activation. In more specificembodiments, the antigen preventing cells are B cells, macrophages ormonocytes.

In one embodiment, the immune response is an autoimmune response. Inspecific embodiments the autoimmune response is selected from the groupconsisting of Type I diabetes, multiple sclerosis, systemic lupuserythematosus, Grave's disease, autoimmune hemolytic anemia, bullouspemphigoid, Hashimoto's thyroiditis, myasthenia gravis, pemphigus,pernicious anemia, and the like.

In another embodiment the immune response is an allogeneic response. Ina specific embodiment, the allogeneic response is selected from thegroup consisting of graft versus host disease and host versus graftdisease.

A third aspect of the invention is a method of suppressing, preventingor ameliorating an inflammatory response in an subject in need thereof,such method comprising administering to the subject an effective amountof a composition selected from the group consisting of a compositioncomprising EHP cells, including AMP cells, conditioned media derivedtherefrom, cell lysates derived therefrom, and cell products derivedtherefrom, each alone or in combination with each other.

A fourth aspect of the invention is a method of ameliorating aninflammatory response in an subject in need thereof, such methodcomprising administering to the subject an effective amount of acomposition selected from the group consisting of a compositioncomprising EHP cells, including AMP cells, conditioned media derivedtherefrom, cell lysates derived therefrom, and cell products derivedtherefrom, each alone or in combination with each other.

In another embodiment the inflammatory response is selected from thegroup consisting of inflammatory diseases of integument, inflammatorybowel diseases and rheumatic diseases. In specific embodiments theinflammatory diseases of the integument are selected from the groupconsisting of psoriasis and atopic dermatitis. In another specificembodiment the inflammatory bowel diseases are selected from the groupconsisting of ulcerative colitis and Crohn's disease. In yet anotherspecific embodiment the rheumatic diseases are selected from the groupconsisting of osteoarthritis, rheumatoid arthritis, juvenile rheumatoidarthritis, fibromyalgia, scleroderma, spondyloarthropathies, gout,infectious arthritis, polymyalgia rheumatica, polymyositis, psoriaticarthritis, bursitis, tendinitis, CIAS1-related Autoinflammatory PeriodicSyndromes (CAPS), pelvic inflammatory disease, interstitial cystitis,Henoh-Schonlein purpura, Behcet's syndrome and the like.

In a preferred embodiment of the invention the subject is a human ornon-human animal.

In another embodiment the composition comprising EHP cells, includingAMP cells, conditioned media derived therefrom, cell lysates derivedtherefrom, or cell products derived therefrom, each alone or incombination with each other, are administered topically, parenterally orenterally. In a preferred embodiment the EHP cells are AMP cells.

In a fifth aspect of the invention, composition of EHP cells, includingAMP cells, conditioned media derived therefrom, cell lysates derivedtherefrom, or cell products derived therefrom, each alone or incombination with each other, is co-administered with one or more activeagents. In one embodiment the active agent is selected from the groupconsisting of corticosteroids, cyclosporine, tacrolimus, sirolimus,methotrexate, azathiopine, mercatopurine, cytotoxic antibiotics,polyclonal antibodies, monoclonal antibodies, interferon, opioids, TNFbinding proteins, mycophenolate, and FTY720. In a specific embodimentthe monoclonal antibodies are selected from the group consisting ofanti-T-cell receptor (CD23) and anti-IL2 receptor (CD25) antibodies

In another embodiment the EHP cells, including AMP cells, conditionedmedia derived therefrom, cell lysates derived therefrom, or cellproducts derived therefrom, are allogeneic to the subject.

A sixth aspect of the invention is a method of preparing suppressorT-cells, wherein the method comprises the steps of contacting activatedeffector T-Cells with allogeneic EHP cells in culture to producesuppressor T-cells and separating the suppressor T-cells from theculture. In one specific embodiment, the EHP cells are AMP cells.

A seventh aspect of the invention is a method of reducing an immuneresponse against an alloantigen, wherein the method comprises the stepsof culturing activated T-cells with EHP cells in vitro, whereby the EHPcells induce the activated T-cells to become suppressor T-cells andcontacting immune effector cells with the suppressor T-cells in anamount effective to reduce the immune response. In one embodiment theEHP cells are AMP cells. In another embodiment, the EHP cells, the Tcells and the effector cells are human cells. In another embodiment, theeffector cells are T-cells.

An eighth aspect of the invention is a method of treating a transplantrecipient for graft versus host disease, wherein the method comprisesthe steps of culturing activated human T-cells with human EHP cells invitro, whereby the human EHP cells induce the activated human T-cells tobecome human suppressor T-cells and treating the recipient of a donortransplant with the human suppressor T-cells in an amount effective toreduce an immune response against the recipient by the transplant. In apreferred embodiment, the EHP cells are AMP cells. In anotherembodiment, the suppressor T-cells are allogeneic to the EHP cells.

A ninth aspect of the invention is a method of reducing an immuneresponse to a donor transplant, wherein the method comprises the stepsof comprising culturing activated human T-cells with human EHP cells invitro to produce human suppressor T-cells and administering to therecipient of the donor transplant the human suppressor T-cells in anamount effective to reduce an immune response in the recipient to thetransplant. In a preferred embodiment the EHP cells are AMP cells. Inanother embodiment, the human EHP cells are allogeneic to the activatedhuman T-cells. In another embodiment the suppressor T-cells areadministered to the recipient prior to administration of the transplant.In another embodiment the suppressor T-cells are administeredconcurrently with administration of the transplant, and in yet anotherembodiment the suppressor T-cells are administered as part of thetransplant, and in still another embodiment the suppressor T-cells areadministered after the transplant.

Other features and advantages of the invention will be apparent from theaccompanying description and the claims. The contents of all references,pending patent applications and published patents, cited throughout thisapplication are hereby expressly incorporated by reference. In case ofconflict, the present specification, including definitions, willcontrol.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Mixed lymphocyte reaction (MLR). Normal peripheral bloodmononuclear cells vs. HLA-DR (Class II) mismatched AMP cells.

FIG. 2: Normal mononuclear cell response to mitogen, MLR, and recallantigen cytomegalovirus (CMV) plus the addition of HLA-DR (Class II)mismatched AMP cells.

FIG. 3: Effects of serially diluted AMP cells on Allo-Antigen MLR.

FIG. 4: Effects of serially diluted AMP cells on memory response tocytomegalovirus (CMV).

DEFINITIONS

As defined herein “isolated” refers to material removed from itsoriginal environment and is thus altered “by the hand of man” from itsnatural state.

As used herein, the term “protein marker” means any protein moleculecharacteristic of the plasma membrane of a cell or in some cases of aspecific cell type.

As used herein, “enriched” means to selectively concentrate or toincrease the amount of one or more materials by elimination of theunwanted materials or selection and separation of desirable materialsfrom a mixture (i.e. separate cells with specific cell markers from aheterogeneous cell population in which not all cells in the populationexpress the marker).

As used herein, the term “substantially purified” means a population ofcells substantially homogeneous for a particular marker or combinationof markers. By substantially homogeneous is meant at least 90%, andpreferably 95% homogeneous for a particular marker or combination ofmarkers.

The term “placenta” as used herein means both preterm and term placenta.

As used herein, the term “totipotent cells” shall have the followingmeaning. In mammals, totipotent cells have the potential to become anycell type in the adult body; any cell type(s) of the extraembryonicmembranes (e.g., placenta). Totipotent cells are the fertilized egg andapproximately the first 4 cells produced by its cleavage.

As used herein, the term “pluripotent stem cells” shall have thefollowing meaning Pluripotent stem cells are true stem cells with thepotential to make any differentiated cell in the body, but cannotcontribute to making the components of the extraembryonic membraneswhich are derived from the trophoblast. The amnion develops from theepiblast, not the trophoblast. Three types of pluripotent stem cellshave been confirmed to date: Embryonic Stem (ES) Cells (may also betotipotent in primates), Embryonic Germ (EG) Cells, and EmbryonicCarcinoma (EC) Cells. These EC cells can be isolated fromteratocarcinomas, a tumor that occasionally occurs in the gonad of afetus. Unlike the other two, they are usually aneuploid.

As used herein, the term “multipotent stem cells” are true stem cellsbut can only differentiate into a limited number of types. For example,the bone marrow contains multipotent stem cells that give rise to allthe cells of the blood but may not be able to differentiate into othercell types.

As used herein, the term “extraembryonic tissue” means tissue locatedoutside the embryonic body which is involved with the embryo'sprotection, nutrition, waste removal, etc. Extraembryonic tissue isdiscarded at birth. Extraembryonic tissue includes but is not limited tothe amnion, chorion (trophoblast and extraembryonic mesoderm includingumbilical cord and vessels), yolk sac, allantois and amniotic fluid(including all components contained therein). Extraembryonic tissue andcells derived therefrom have the same genotype as the developing embryo.

As used herein, the term “extraembryonic cells” or “EE cells” means apopulation of cells derived from the extraembryonic tissue.

As used herein, the term “EHP cells” means a population of cells derivedfrom the extraembryonic tissue which have the characteristics of beingHLA-G positive upon isolation, are MHC Class II negative, do not expressthe co-stimulatory molecules CD80 and CD86 and are not MAPCs asdescribed in US Published Patent Application No. 20060263337.

As used herein, the term “amnion-derived multipotent progenitor cell” or“AMP cell” means a population of epithelial cells that are derived fromthe amnion. In addition to the characteristics described above for EHPcells, AMP cells grow without feeder layers, do not express the proteintelomerase and are non-tumorigenic. AMP cells do not express thehematopoietic stem cell marker CD34 protein. The absence of CD34positive cells in this population indicates the isolates are notcontaminated with hematopoietic stem cells such as umbilical cord bloodor embryonic fibroblasts. Virtually 100% of the cells react withantibodies to low molecular weight cytokeratins, confirming theirepithelial nature. Freshly isolated AMP cells will not react withantibodies to the stem/progenitor cell markers c-kit and Thy-1. Severalprocedures used to obtain cells from full term or pre-term placenta areknown in the art (see, for example, US 2004/0110287; Anker et al., 2005,Stem Cells 22:1338-1345; Ramkumar et al., 1995, Am. J. Ob. Gyn.172:493-500). However, the methods used herein provide improvedcompositions and populations of cells. AMP cells have previously beendescribed as “amnion-derived cells” (see U.S. Provisional ApplicationNos. 60/666,949, 60/699,257, 60/742,067, U.S. Provisional ApplicationNos. 60/813,759, U.S. application Ser. No. 11/333,849, U.S. applicationSer. No. 11/392,892, and PCTUS06/011392, each of which is incorporatedherein in its entirety).

The term “composition of extraembryonic cells” as used herein includesthe cells and compositions described in this application and inUS2003/0235563, US2004/0161419, US2005/0124003, U.S. ProvisionalApplication Nos. 60/666,949, 60/699,257, 60/742,067, 60/813,759, U.S.application Ser. No. 11/333,849, U.S. application Ser. No. 11/392,892,PCTUS06/011392, US2006/0078993, PCT/US00/40052, U.S. Pat. No. 7,045,148,US2004/0048372, and US2003/0032179, the contents of which areincorporated herein by reference in their entirety.

By the term “animal-free” when referring to compositions, growthconditions, culture media, etc. described herein, is meant that noanimal-derived materials, such as animal-derived serum, other than humanmaterials, such as native or recombinantly produced human proteins, areused in the preparation, growth, culturing, expansion, or formulation ofthe composition or process.

By the term “expanded”, in reference to EHP cell compositions, meansthat the EHP cell population constitutes a significantly higherconcentration of cells than is obtained using previous methods. Forexample, the level of cells per gram of amniotic tissue in expandedcompositions of AMP cells is at least 50 and up to 150 fold higher thanthe number of cells in the primary culture after 5 passages, as comparedto about a 20 fold increase in such cells using previous methods. Inanother example, the level of cells per gram of amniotic tissue inexpanded compositions of AMP cells is at least 30 and up to 100 foldhigher than the number of cells in the primary culture after 3 passages.Accordingly, an “expanded” population has at least a 2 fold, and up to a10 fold, improvement in cell numbers per gram of amniotic tissue overprevious methods. The term “expanded” is meant to cover only thosesituations in which a person has intervened to elevate the number of theEHP cells. As used herein “passage” or “passaging” refers tosubculturing of cells. For example, cells isolated from the amnion arereferred to as primary cells. Such cells are expanded in culture bybeing grown in the growth medium described herein. When such primarycells are subcultured, each round of subculturing is referred to as apassage. As used herein, “primary culture” means the freshly isolatedEHP cell population.

As used herein, “conditioned medium” is a medium in which a specificcell or population of cells has been cultured, and then removed. Whencells are cultured in a medium, they may secrete cellular factors thatcan provide support to or affect the behavior of other cells. Suchfactors include, but are not limited to hormones, cytokines,extracellular matrix (ECM), proteins, vesicles, antibodies, chemokines,receptors, inhibitors and granules. The medium containing the cellularfactors is the conditioned medium. Examples of methods of preparingconditioned media are described in U.S. Pat. No. 6,372,494 which isincorporated by reference in its entirety herein. As used herein,conditioned medium also refers to components, such as proteins, that arerecovered and/or purified from conditioned medium or from EHP cellsincluding AMP cells.

As used herein, the term “amnion-derived cellular cytokine solution” or“ACCS” means conditioned medium that has been derived from AMP cells orexpanded AMP cells. ACCS has previously been referred to as“amnion-derived cellular cytokine suspension”.

As used herein, “specific activity” means the specific activity of EHPcells, including AMP cells, and is determined by calculating a 50%inhibition dosage (ID₅₀). For example, using a standardallogeneic-antigen MLR, the 100% response is calculated by determiningthe PBMC responder response to the mismatched stimulator withoutaddition of AMP cells. Then, AMP cells are titered into the MLR at 1:2serial dilutions. The number of AMP cells required to half the 100%response is reported as the ID₅₀.

The term “lysate” as used herein refers to the composition obtained whencells, for example, EHP cells, are lysed and optionally the cellulardebris (e.g., cellular membranes) is removed. This may be achieved bymechanical means, by freezing and thawing, by use of detergents, such asEDTA, or by enzymatic digestion using, for example, hyaluronidase,dispase, proteases, and nucleases.

As used herein, the term “substrate” means a defined coating on asurface that cells attach to, grown on, and/or migrate on. As usedherein, the term “matrix” means a substance that cells grow in or onthat may or may not be defined in its components. The matrix includesboth biological and non-biological substances. As used herein, the term“scaffold” means a three-dimensional (3D) structure (substrate and/ormatrix) that cells grow in or on. It may be composed of biologicalcomponents, synthetic components or a combination of both. Further, itmay be naturally constructed by cells or artificially constructed. Inaddition, the scaffold may contain components that have biologicalactivity under appropriate conditions.

The term “cell product” or “cell products” as used herein refers to anyand all substances made by and secreted from a cell, including but notlimited to, protein factors (i.e. growth factors, differentiationfactors, engraftment factors, cytokines, morphogens, proteases (i.e. topromote endogenous cell delamination, protease inhibitors),extracellular matrix components (i.e. fibronectin, etc.).

The term “transplantation” refers to the administration of a compositioneither in an undifferentiated, partially differentiated, or fullydifferentiated form into a human or other animal.

As used herein, the terms “a” or “an” means one or more; at least one.

As used herein, the term “adjunctive” means jointly, together with, inaddition to, in conjunction with, and the like.

As used herein, the term “co-administer” can include simultaneous orsequential administration of two or more agents. As used herein, theterm “syngeneic” means genetically identical members of the samespecies.

As used herein, the term “allogeneic” means variation in alleles amongmembers of the same species.

As used herein, the terms “immunosuppressive drugs” or“immunosuppressants” are drugs that are used in immunosuppressivetherapy to inhibit or prevent activity of the immune system.

As used herein, the term “GVHD” refers to graft versus host disease,which means the processes that occur primarily in an immunocompromisedhost when it is recognized as non-self by immunocompetent cells of agraft.

As used herein, the term “HVG” refers to host versus graft response,which means the processes which occur when a host rejects a graft.Typically, HVG is triggered when a graft is recognized as foreign(non-self) by immunocompetent cells of the host.

As used herein, the terms “inflammation” or “inflammatory response”means the reaction that occurs in the affected cells and adjacenttissues in response to an injury or abnormal stimulation caused by aphysical, chemical, or biologic substance.

As used herein, the term “immune response” means the cells, tissues andprotein factors (i.e. cytokines) involved in recognizing and attackingforeign substances within the body of an animal.

As used herein, the term “pharmaceutically acceptable” means that thecomponents, in addition to the therapeutic agent, comprising theformulation, are suitable for administration to the patient beingtreated in accordance with the present invention.

The terms “parenteral administration” and “administered parenterally”are art-recognized and refer to modes of administration other thanenteral and topical administration, usually by injection, and includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intra-articular, subcapsular, subarachnoid, intraspinal, andintrasternal injection and infusion.

As used herein “subject” may mean either a human or non-human animal.

As used herein, the term “tissue” refers to an aggregation of similarlyspecialized cells united in the performance of a particular function.

As used herein, the term “therapeutic protein” includes a wide range ofbiologically active proteins including, but not limited to, growthfactors, enzymes, hormones, cytokines, inhibitors of cytokines, bloodclotting factors, peptide growth and differentiation factors.

“Treatment,” “treat,” or “treating,” as used herein covers any treatmentof a disease or condition of a mammal, particularly a human, andincludes: (a) preventing the disease or condition from occurring in asubject which may be predisposed to the disease or condition but has notyet been diagnosed as having it; (b) inhibiting the disease orcondition, i.e., arresting its development; (c) relieving and orameliorating the disease or condition, i.e., causing regression of thedisease or condition; or (d) curing the disease or condition, i.e.,stopping its development or progression. The population of subjectstreated by the methods of the invention includes subjects suffering fromthe undesirable condition or disease, as well as subjects at risk fordevelopment of the condition or disease.

DETAILED DESCRIPTION

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, 2001, “MolecularCloning: A Laboratory Manual”.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and” and “the” include plural references unless thecontext clearly dictates otherwise.

Therapeutic Applications

It has been found that relatively small amounts of EHP cells cansuppress inflammatory and immune responses. Accordingly, in certainaspects of the invention, certain embodiments provide compositions andmethods for treating, ameliorating, and/or preventing or eliminating,adverse immune responses, such as those that occur in transplantationtherapies. The low immunogenicity of allogeneic EHP cells, their abilityto suppress adverse inflammatory and immune responses, and their highspecific activity (as defined in the definitions and elsewhere herein)makes them particularly valuable for adjunctive therapies in thetreatment of such diseases. EHP cells also are useful asimmunosuppressive adjunctive therapeutics for treating adverse immuneresponses that occur in transplantation therapy (i.e. HVG and GVHD).Other immune disorders and diseases are discussed in more detailelsewhere herein. EHP cells further can be useful in adjunctiveimmunosuppressive therapy in the treatment of certain inflammatorydiseases. Such diseases are discussed in greater detail below andelsewhere in the specification. It should be noted that the diseases andconditions discussed below are intended to be examples of suitablediseases and conditions which may be treated by the methods of theinvention. Skilled artisans will recognize that treatment of many otherrelated diseases and conditions are also contemplated by the methods ofthe invention.

Using the methods described herein for EHP cell isolation,characterization, and expansion, together with the disclosure herein onthe immune-suppressing properties of EHP cells, EHP cells can be used toprevent, suppress, or diminish immune disorders, dysfunctions, ordiseases, including, for example, adverse immune reactions, such asthose that result from other therapies, including those that complicatetransplantation therapies, such as HVG and GVHD. Such disorders,dysfunctions, and diseases also include congenital immune disorders andautoimmune diseases, among others. EHP cells are useful as both aprimary and adjunctive therapeutic agent. EHP cells can be usedtherapeutically alone or together with other agents; can be administeredbefore, during, and/or after such agents; used alone or with otheragents; can be administered before, during, and/or after a transplant.If administered during transplant, EHP cells can be administeredtogether with the transplant material or separately. If separatelyadministered, the EHP cells can be administered sequentially orsimultaneously with the transplant. Furthermore, EHP cells may beadministered in advance of the transplant and/or after the transplant.Other agents that can be used in conjunction with EHP cells, intransplantation therapies in particular, include immunomodulatoryagents. A variety of such agents are described elsewhere herein. Incertain embodiments of the invention, the immunomodulatory agents areimmunosuppressive agents, such as those described elsewhere herein.

In certain embodiments the EHP cells are administered by injection, suchas by intravenous injection; are encapsulated for administration; areadministered in situ (i.e. solid organ transplantation and organrepair). These and other forms of administration are discussed in detailelsewhere herein. In some embodiments of the invention, EHP cells areadministered in doses measured by the ratio of EHP cells (cells) to bodymass (weight). Alternatively, they can be administered in doses of afixed number of cells. Dosing, routes of administration, formulations,and the like are discussed in greater detail elsewhere herein.

Because of their novel immunomodulatory properties, EHP cells, includingAMP cells, conditioned media derived therefrom, cell lysates derivedtherefrom, or cell products derived therefrom, each alone or incombination, or in combination with other active agents, areparticularly useful in the prevention and/or the amelioration ofinflammatory and/or immune responses and/or allergic reactions.Inflammatory responses are characterized by dilation of blood vessels inthe affected area resulting in increased blood flow to the area. If atthe surface, it makes skin look red and feel warm. Capillaries in thearea become more permeable allowing fluid to seep into the surroundingtissue resulting in edematous swelling around the infected site. Theswelling and the effects of some of the chemicals released results inpain. Hence clinical characteristics of the inflammatory response areknown as redness, heat, edema and pain. The inflammatory response occursas a result of chemical messages produced by “mediators”. There are twomajor classes of mediators: 1) cell-derived mediators which are producedby leukocytes and 2) plasma-derived mediators which are found in theblood plasma. Cell-derived mediators include arachidonic acidderivatives (i.e. prostaglandins and leukotrines), cytokines,lymphokines and monokines, interleukin, platelet activating factor(PAF), histamine and bradykinin. Plasma-derived mediators includecomplement and interferons. Inflammatory responses may be genetic innature (i.e. CAPS, see below for details). They may be environmentallyinduced or virally induced. Non-limiting examples of diseases havinginflammatory responses which are suitable for treatment with EHP cells,including AMP cells, conditioned media derived therefrom, cell lysatesderived therefrom, or cell products derived therefrom, each alone or incombination, including in combination with other active agents, includethe following:

Inflammatory Diseases of Integument which include psoriasis and eczema(i.e. atopic dermatitis). Psoriasis is an inflammatory skin condition.There are five types, each with unique signs and symptoms. Between 10%and 30% of people who develop psoriasis get a related form of arthritiscalled “psoriatic arthritis,” which causes inflammation of the joints.Plaque psoriasis is the most common type of psoriasis. About 80% ofpeople who develop psoriasis have plaque psoriasis, which appears aspatches of raised, reddish skin covered by silvery-white scale. Thesepatches, or plaques, frequently form on the elbows, knees, lower back,and scalp. However, they can occur anywhere on the body. The other typesare guttate psoriasis (small, red spots on the skin), pustular psoriasis(white pustules surrounded by red skin), inverse psoriasis (smooth, redlesions form in skin folds), and erythrodermic psoriasis (widespreadredness, severe itching, and pain). Regardless of type, psoriasisusually causes discomfort. The skin often itches, and it may crack andbleed. In severe cases, the itching and discomfort may keep a personawake at night, and the pain can make everyday tasks difficult.Psoriasis is a chronic, meaning lifelong, condition because there iscurrently no cure. People often experience flares and remissionsthroughout their life. Controlling the signs and symptoms typicallyrequires lifelong therapy. Eczema is a general term encompassing variousinflamed skin conditions. One of the most common forms of eczema isatopic dermatitis. Approximately 10-20% of the world population isaffected by this chronic, relapsing, and very itchy rash at some pointduring childhood. Fortunately, many children with eczema find that thedisease clears and often disappears with age. In general, atopicdermatitis will come and go, often based on external factors. Althoughits cause is unknown, the condition appears to be an abnormal responseof the body's immune system. In people with eczema, the inflammatoryresponse to irritating substances overacts, causing itching andscratching. Eczema is not contagious and, like many diseases, currentlycannot be cured. However, for most patients the condition may be managedwell with treatment and avoidance of triggers.

Inflammatory Bowel Diseases (IBDs) are chronic inflammatory diseases ofthe GI tract of unknown etiology, and include ulcerative colitis (UC)and Crohn's disease (CD), which is also referred to as regionalenteritis, terminal ileitis, or granulomatous ileocolitis. Ulcerativecolitis is an inflammatory disease of the large intestine, also calledthe colon. In ulcerative colitis, the inner lining (mucosa) of theintestine becomes inflamed and develops ulcers (an ulcer is a sore,which means it's an open, painful wound). Ulcerative colitis is oftenthe most severe in the rectal area, which can cause frequent diarrhea.Mucus and blood often appear in the stool if the lining of the colon isdamaged. Crohn's disease differs from ulcerative colitis in the areas ofthe bowel it involves—it most commonly affects the last part of thesmall intestine called the terminal ileum and parts of the largeintestine. However, Crohn's disease isn't limited to these areas and canattack any part of the digestive tract. Crohn's disease causesinflammation that extends much deeper into the layers of the intestinalwall than ulcerative colitis does. Crohn's disease generally tends toinvolve the entire bowel wall, whereas ulcerative colitis affects onlythe lining of the bowel.

Rheumatic Diseases Encompass Many Diseases Including:

Osteoarthritis—This is the most common type of arthritis, affecting anestimated 21 million adults in the United States. Osteoarthritisprimarily affects cartilage, which is the tissue that cushions the endsof bones within the joint. In osteoarthritis, the cartilage begins tofray and may entirely wear away. Osteoarthritis can cause joint pain andstiffness. Disability results most often when the disease affects thespine and the weight-bearing joints (the knees and hips).

Rheumatoid arthritis—This inflammatory disease of the synovium, orlining of the joint, results in pain, stiffness, swelling, joint damage,and loss of function of the joints. Inflammation most often affectsjoints of the hands and feet and tends to be symmetrical (occurringequally on both sides of the body). This symmetry helps distinguishrheumatoid arthritis from other forms of the disease. About 1 percent ofthe U.S. population (about 2.1 million people) has rheumatoid arthritis.

Juvenile rheumatoid arthritis—This is the most common form of arthritisin childhood, causing pain, stiffness, swelling, and loss of function ofthe joints. The arthritis may be associated with rashes or fevers, andmay affect various parts of the body.

Fibromyalgia—Fibromyalgia is a chronic disorder that causes painthroughout the tissues that support and move the bones and joints. Pain,stiffness, and localized tender points occur in the muscles and tendons,particularly those of the neck, spine, shoulders, and hips. Patients mayalso experience fatigue and sleep disturbances.

Scleroderma—Also known as systemic sclerosis, scleroderma meansliterally “hard skin.” The disease affects the skin, blood vessels, andjoints. It may also affect internal organs, such as the lungs andkidneys. In scleroderma, there is an abnormal and excessive productionof collagen (a fiber-like protein) in the skin or internal organs.

Spondyloarthropathies—This group of rheumatic diseases principallyaffects the spine. One common form—ankylosing spondylitis—not onlyaffects the spine, but may also affect the hips, shoulders, and knees asthe tendons and ligaments around the bones and joints become inflamed,resulting in pain and stiffness. Ankylosing spondylitis tends to affectpeople in late adolescence or early adulthood. Reactive arthritis,sometimes called Reiter's syndrome, is another spondyloarthropathy. Itdevelops after an infection involving the lower urinary tract, bowel, orother organ and is commonly associated with eye problems, skin rashes,and mouth sores.

Gout—This type of arthritis results from deposits of needle-likecrystals of uric acid in the joints. The crystals cause inflammation,swelling, and pain in the affected joint, which is often the big toe.

Infectious arthritis—This is a general term used to describe forms ofarthritis that are caused by infectious agents, such as bacteria orviruses. Parvovirus arthritis and gonococcal arthritis are examples ofinfectious arthritis. Arthritis symptoms may also occur in Lyme disease,which is caused by a bacterial infection following the bite of certainticks. In those cases of arthritis caused by bacteria, early diagnosisand treatment with antibiotics are crucial to get rid of the infectionand minimize damage to the joints.

Polymyalgia rheumatica—Because this disease involves tendons, muscles,ligaments, and tissues around the joint, symptoms often include pain,aching, and morning stiffness in the shoulders, hips, neck, and lowerback. It is sometimes the first sign of giant cell arteritis, a diseaseof the arteries characterized by inflammation, weakness, weight loss,and fever.

Polymyositis—This is a rheumatic disease that causes inflammation andweakness in the muscles. The disease may affect the whole body and causedisability.

Psoriatic arthritis—This form of arthritis occurs in some patients withpsoriasis, a scaling skin disorder. Psoriatic arthritis often affectsthe joints at the ends of the fingers and toes and is accompanied bychanges in the fingernails and toenails. Back pain may occur if thespine is involved.

Bursitis—This condition involves inflammation of the bursae, small,fluid-filled sacs that help reduce friction between bones and othermoving structures in the joints. The inflammation may result fromarthritis in the joint or injury or infection of the bursae. Bursitisproduces pain and tenderness and may limit the movement of nearbyjoints.

Tendinitis (Tendonitis)—This condition refers to inflammation of tendons(tough cords of tissue that connect muscle to bone) caused by overuse,injury, or a rheumatic condition. Tendinitis produces pain andtenderness and may restrict movement of nearby joints.

Pelvic inflammatory disease (PID)—Is a general term that refers toinfection and inflammation of the upper genital tract in women. It canaffect the uterus (womb), fallopian tubes (tubes that carry eggs fromthe ovaries to the uterus), ovaries, and other organs related toreproduction. The scarring that results on these organs can lead toinfertility, tubal (ectopic) pregnancy, chronic pelvic pain, abscesses(sores containing pus), and other serious problems. PID is the mostcommon preventable cause of infertility in the United States.

CIAS1-related Autoinflammatory Periodic Syndromes (CAPS)— is a spectrumof rare inherited inflammatory conditions, including Familial ColdAutoinflammatory Syndrome (FCAS), Muckle-Wells Syndrome (MWS), andNeonatal Onset Multisystem Inflammatory Disease (NOMID). These syndromesare characterized by spontaneous systemic inflammation and are termedautoinflammatory disorders. A novel feature of these conditions(particularly FCAS and MWS) is that exposure to mild degrees of coldtemperature can provoke a major inflammatory episode that occurs withinhours. CAPS are caused by a range of mutations in the gene CIAS1 (alsoknown as NALP3)

Miscellaneous inflammatory diseases include interstitial cystitis,Henoh-Schonlein purpura, and Behcet's syndrome.

Non-limiting examples of diseases having immune responses which aresuitable for treatment with EHP cells, including AMP cells, conditionedmedia derived therefrom, cell lysates derived therefrom, or cellproducts derived therefrom, each alone or in combination, including incombination with other active agents, include:

GVHD refers to graft versus host disease, which means the processes thatoccur primarily in an immunocompromised host when it is recognized asnon-self by immunocompetent cells of a graft.

HVG refers to host versus graft response, which means the processeswhich occur when a host rejects a graft. Typically, HVG is triggeredwhen a graft is recognized as foreign (non-self) by immunocompetentcells of the host.

Juvenile (Type I) Diabetes is caused when the body forms antibodies thatattack the beta cells in the islets of Langerhans in the pancreas. Sincethe beta cells are responsible for producing insulin, Type I diabeticsmust inject themselves with insulin their entire life.

Systemic lupus erythematosus (also known as lupus or SLE) is anautoimmune disease in which the immune system harms the body's ownhealthy cells and tissues. This can result in inflammation of and damageto the joints, skin, kidneys, heart, lungs, blood vessels, and brain.

Graves' disease is caused by an abnormal immune system response thatattacks the thyroid gland, and causes too much production of thyroidhormones. Risk factors are being a woman over 20 years old, although thedisorder may occur at any age and may affect men as well.

Miscellaneous immune disorders include autoimmune hemolytic anemia,bullous pemphigoid, Hashimoto's thyroiditis, myasthenia gravis,pemphigus, and pernicious anemia.

Allergic reactions are sensitivities to a specific substance, called anallergen, which is contacted through the skin, inhaled into the lungs,swallowed, or injected. Allergic reactions vary. They can be mild orserious. They can be confined to a small area of the body or may affectthe entire body. Most occur within seconds or minutes after exposure tothe allergen, but some can occur after several hours, particularly ifthe allergen causes a reaction after it is partially digested. In veryrare cases, reactions develop after 24 hours. Anaphylaxis is a suddenand severe allergic reaction that occurs within minutes of exposure.Immediate medical attention is needed for this condition. It can getworse very, very fast and lead to death within 15 minutes if treatmentis not received.

General Approaches for Transplantation/Cell-Based Therapies

One of skill in the art will recognize that HLA-G expression by cells isimportant in a cell's ability to evade immunosurveillance. Therefore,any cell that expresses HLA-G is potentially useful in practicing themethods of the invention. So, in addition to EHP cells and AMP cells,this includes cells such as certain tumor cells (see, for example,Tripathi, P. and Agrawal, S., Cancer Invest 2006, 24(2):178-186;Blaschitz, A., et al., Human Immunology 2000, 61:1074-1085), certaincells in the thymus (see, for example, Mallet, V., et al., Int Immunol1999, 11:889-898; Mallet, V., et al., Reprod Immunol 1999,43(2):225-234) and non-progenitor placental-derived HLA-G positive cells(Blaschitz, A., et al., Human Immunology 2000, 61:1074-1085).

Isolation, Identification and Characterization of EHP Cells

Various methods for isolating cells from the extraembryonic tissue,which may then be used to produce the EHP cells of the instant inventionare described in the art (see, for example, US2003/0235563,US2004/0161419, US2005/0124003, U.S. Provisional Application Nos.60/666,949, 60/699,257, 60/742,067, 60/813,759, U.S. application Ser.No. 11/333,849, U.S. application Ser. No. 11/392,892, PCTUS06/011392,US2006/0078993, PCT/US00/40052, U.S. Pat. No. 7,045,148, US2004/0048372,and US2003/0032179).

Identifying EHP cells—Once EE cells are isolated, it is necessary toidentify which cells have the characteristics associated with EHP cells.For example, the cells are tested for the presence of HLA-G, the absenceof MHC Class II antigens, and the absence of co-stimulatory moleculesCD80 and CD86.

Isolation, Identification and Characterization of AMP Cells

In accordance with the invention, AMP cell compositions are preparedusing the steps of a) recovery of the amnion from the placenta, b)dissociation of the cells from the amniotic membrane, c) culturing ofthe cells in a basal medium with the addition of a naturally derived orrecombinantly produced human protein; and optionally d) furtherproliferation of the cells using additional additives and/or growthfactors. Details are contained in US Publication No. 2006-0222634-A1,which is incorporated herein by reference.

The AMP cells of the invention are characterized as follows: Usingcommercially available antibodies to known stem cell markers, freshlyisolated AMP cells have been extensively characterized. Briefly, freshlyisolated AMP cells are substantially negative with respect to CD90 andCD117. In addition, such cell populations are essentially negative forprotein expression of CD34, CD44, CD45, CD140b, CD105; essentiallypositive for protein expression of CD9 and CD29; between about 70-95%positive for protein expression of SSEA4, CD10, CD166 and CD227; betweenabout 60-95% positive for protein expression of HLA-G, EGFR and CD26;and between about 10-50% positive for protein expression of CD71.Details on this procedure are contained in US Publication No.2006-0222634-A1, which is incorporated herein by reference.

In alternative embodiments substantially purified AMP cell populationscan be created using antibodies against protein markers expressed(positive selection) or not expressed (negative selection) on the cellsurface of the AMP cells. These antibodies may be used to identify,characterize, isolate or create such substantially purified populationsof AMP cells expressing those protein markers using a variety ofmethods. Details on this procedure are contained in US Publication No.2006-0222634-A1, which is incorporated herein by reference.

In addition, protein markers that are not expressed on the surface ofAMP cells may also be used to identify, isolate or create populations ofAMP cells not expressing those markers. Such procedures may involve anegative selection method, such as passage of sample cells over a columncontaining anti-protein marker antibodies or by binding of cells tomagnetic bead-conjugated antibodies to the protein markers or by panningon plates coated with protein marker antibodies and collecting theunbound cells. Alternatively, a single-cell suspension may be exposed toone or more fluorescent-labeled antibodies that immuno-specifically bindto the protein markers. Details on this procedure are contained in USPublication No. 2006-0222634-A1, which is incorporated herein byreference.

Expanded Populations of EHP Cells, Including AMP Cells

One of skill in the art will recognize that any of the EHP cells of theinstant invention may be expanded using the methods described below forAMP cells.

As described herein and in US Publication No. 2006-0222634-A1, which isincorporated herein by reference, Applicants have discovered a novelmethod for isolation and propagation of multipotent AMP cells. Suchmethods result in AMP cell compositions which are expanded formultipotent cells, thereby providing, for the first time, sufficientquantities of cells to enable therapeutic cell transplantation. ExpandedAMP cell compositions, which are made in accordance with the subjectinvention, are compositions in which the level of cells per gram ofamniotic tissue is at least 50 fold and up to 150 fold higher after 5passages, as compared to about 20 fold using previous methods.Alternatively, expanded AMP cell compositions, which are made inaccordance with the subject invention, are compositions in which thelevel of cells per gram of amniotic tissue is at least 30 fold and up to100 fold higher after 3 passages.

Additionally, the methods used for cell culture and proliferationprovide a means to culture the cells, as well as other cells includingbut not limited to multipotent, pluripotent cells or totipotent cells,including, but not limited to, embryonic stem cells, in an animal-freesystem. Furthermore, the culture conditions described provide a cellthat is less dependent on attachment to a culture surface for viability,thus allowing for propagation of the cells using suspension culture forefficient scale-up. Details on this procedure are contained in USPublication No. 2006-0222634-A1, which is incorporated herein byreference.

The expanded AMP cell compositions described herein demonstrateextensive proliferative potential, express certain genes known to beexpressed only in undifferentiated cells (i.e. Nanog and Oct-4) and candifferentiate into cell types that normally arise from all threeembryonic germ layers (endoderm, ectoderm and mesoderm). Thisdifferentiation potential suggests that these expanded AMP cells may beable to contribute to a variety of cell types. The AMP cell compositionsdescribed herein are also useful as feeder layers for the growth of avariety of cell types, including but not limited to embryonic stem cells(ES cells). AMP cells, including those described herein, also produce awide variety of cytokines and growth factors, thereby making both thecell compositions, conditioned media derived from the cells (ACCS), celllysates therefrom, extracellular matrices produced by the cells, andcombinations thereof useful for a variety of therapeutic applications,in particular cell-based therapeutic applications such astransplantation therapies.

In addition, the novel immunomodulating properties possessed by thecompositions of the invention and described herein make the compositionsuseful in clinical settings in which modulating inflammatory and/orimmune responses and/or treating, preventing and/or amelioratinginflammatory, immune, and/or allergic diseases and disorders in asubject in need thereof is indicated. Such uses are accomplished byadministering EHP cells, including AMP cells, conditioned media derivedtherefrom, cell lysates derived therefrom, and cell products derivedtherefrom, each alone or in combination, including in combination witheach other and/or other suitable active agents. Further details on thetherapeutic potential of these cells and cell products are contained inUS Publication No. 2006-0222634-A1, which is incorporated herein byreference.

Compositions—The compositions of the invention include substantiallypurified populations of EHP cells, including AMP cells, as well as celllysates and/or conditioned media derived therefrom, alone or incombination with each other and/or other suitable active agents, andpharmaceutical compositions of such. The compositions of the inventioncan be prepared in a variety of ways depending on the intended use ofthe compositions. For example, a composition useful in practicing theinvention may be a liquid comprising an agent of the invention, i.e. asubstantially purified population of EHP cells, in solution, insuspension, or both (solution/suspension). The term“solution/suspension” refers to a liquid composition where a firstportion of the active agent is present in solution and a second portionof the active agent is present in particulate form, in suspension in aliquid matrix. A liquid composition also includes a gel. The liquidcomposition may be aqueous or in the form of an ointment, salve, cream,or the like.

An aqueous suspension or solution/suspension useful for practicing themethods of the invention may contain one or more polymers as suspendingagents. Useful polymers include water-soluble polymers such ascellulosic polymers and water-insoluble polymers such as cross-linkedcarboxyl-containing polymers. An aqueous suspension orsolution/suspension of the present invention is preferably viscous ormuco-adhesive, or even more preferably, both viscous and muco-adhesive.Furthermore, the compositions may be combined with one or more activeagents. Such other active agents include but are not limited toimmunosuppressive agents such as cyclosporine, methotrexate, FK-506,corticosteroids and the like (see below).

Pharmaceutical Compositions

The present invention provides pharmaceutical compositions ofsubstantially purified populations of EHP cells, including AMP cells, aswell as cell lysates and/or conditioned media derived therefrom, aloneor in combination with each other and/or other suitable active agents,and a pharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly, inhumans. The term “carrier” refers to a diluent, adjuvant, excipient, orvehicle with which the composition is administered. Such pharmaceuticalcarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Suitablepharmaceutical excipients include starch, glucose, lactose, sucrose,gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. Examples of suitablepharmaceutical carriers are described in “Remington's PharmaceuticalSciences” by E. W. Martin, and still others are familiar to skilledartisans.

The pharmaceutical compositions of the invention can be formulated asneutral or salt forms. Pharmaceutically acceptable salts include thoseformed with free amino groups such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withfree carboxyl groups such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc. Furthermore, thepharmaceuticals compositions may be combined with one or more activeagents. Such other active agents include but are not limited toimmunosuppressive agents such as cyclosporine, methotrexate, FK-506,corticosteroids and the like (see below).

Treatment Kits

The invention also provides for an article of manufacture comprisingpackaging material and a pharmaceutical composition of the inventioncontained within the packaging material, wherein the pharmaceuticalcomposition comprises a substantially purified population of EHP cells,including AMP cells, as well cell lysates and/or conditioned mediaderived therefrom, alone or in combination with each other, andoptionally in combination with other active agents and wherein thepackaging material comprises a label or package insert which indicatesthat the substantially purified population of EHP cells including AMPcells, as well cell lysates and/or conditioned media derived therefrom,alone or in combination with each other can be used for treating avariety of disorders including but not limited to HVG, GVHD, and otherinflammatory and immune diseases and disorders, allergic reactions, etc.

EHP cells, including AMP cells, as well cell lysates and/or conditionedmedia derived therefrom, alone or in combination with each other, areuseful both as primary and adjunctive therapeutic agents and modalities.Such compositions can be used therapeutically alone or together withother agents. Such compositions can be administered before, during,and/or after such agents. Likewise, whether used alone or with otheragents, such compositions can be administered before, during, and/orafter a transplant or other cell-based therapy. If administered duringtransplant or other cell-based therapy, the compositions can beadministered together with the transplant or other cell-based therapymaterial, or administered separately. If separately administered, thecompositions can be administered sequentially or simultaneously with thetransplant or other cell-based therapy. Furthermore, the compositionsmay be administered in advance of the transplant or other cell-basedtherapy and/or after the transplant or other cell-based therapy. Inaddition, the compositions maybe administered as the transplant. In suchsituations, they may be administered alone or in combination with otheractive agents, etc.

Other Agents Useful for Co-Administration.

EHP cells, including AMP cells, conditioned media derived therefrom,cell lysates derived therefrom, or cell products derived therefrom, eachalone or in combination may be administered with other pharmaceuticallyactive agents. In some embodiments one or more of such agents areformulated together with the compositions for administration. In someother embodiments the compositions and the one or more agents are inseparate formulations. In yet some other embodiments the compositionscomprising the EHP cells, including AMP cells, conditioned media derivedtherefrom, cell lysates derived therefrom, or cell products derivedtherefrom, each alone or in combination and/or the one or more agentsare formulated with regard to adjunctive use with one another.

EHP cells, including AMP cells, conditioned media derived therefrom,cell lysates derived therefrom, or cell products derived therefrom, eachalone or in combination, may be administered in a formulation comprisinga immunosuppressive agents, such as any combination of any number ofcorticosteroids (i.e. glucocorticoids), cyclosporine, tacrolimus,sirolimus, methotrexate, azathiopine, mercatopurine, cytotoxicantibiotics, polyclonal and monoclonal antibodies such as anti-T-cellreceptor (CD23) and anti-IL2 receptor (CD25) antibodies, interferon,opioids, TNF binding proteins, mycophenolate, and small biologicalagents such as FTY720. Immunosuppressive agents in accordance with theforegoing may be the only such additional agents or may be combined withother agents.

Such agents also include antibiotic agents, antifungal agents, andantiviral agents, to name just a few other pharmacologically activesubstances and compositions that may be used in accordance withembodiments of the invention. Typical antibiotics or anti-mycoticcompounds include, but are not limited to, penicillin, streptomycin,amphotericin, ampicillin, gentamicin, kanamycin, mycophenolic acid,nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin,rifampicin, spectinomycin, tetracycline, tylosin, zeocin, andcephalosporins, aminoglycosides, and echinocandins.

Additional agents may be used in conjunction with the EHP cells,including AMP cells of the present invention, to target the cells to aparticular site, such as one of immune disorder, dysfunction, ordisease, where they might be needed. Such agents may include growthfactors and trophic signaling agents, such as cytokines. They may beused to attract EHP cells to therapeutically targeted sites. They may beadministered to a subject prior to treatment with EHP cells, togetherwith EHP cells, or after EHP cells are administered. Certain cytokines,for instance, alter or affect the migration of EHP cells or cellsdifferentiated therefrom to sites in need of therapy, such asimmunocompromised sites. Cytokines that may be used include, but are notlimited to, stromal cell derived factor-1 (SDF-1), stem cell factor(SCF), angiopoietin-1, placenta-derived growth factor (PlGF),granulocyte-colony stimulating factor (G-CSF), cytokines that stimulateexpression of endothelial adhesion molecules such as ICAMs and VCAMs,and cytokines that engender or facilitate targeting. They may beadministered to a subject as a pre-treatment, along with EHP cells,including AMP cells, or after the compositions have been administered,to promote migration to desired sites and to achieve improvedtherapeutic effect. Such factors may be combined with EHP cells,including AMP cells, alone or in combination in a formulation suitablefor them to be administered together. Alternatively, such factors may beformulated and administered separately.

Order of administration, formulations, doses, frequency of dosing, androutes of administration of factors (such as the cytokines) and EHPcells, including AMP cells, conditioned media derived therefrom, celllysates derived therefrom, or cell products derived therefrom, eachalone or in combination, generally will vary with the disorder ordisease being treated, its severity, the subject, other therapies thatare being administered, the stage of the disorder or disease, andprognostic factors, among others. General regimens that have beenestablished for other treatments provide a framework for determiningappropriate dosing in EHP cell-mediated direct or adjunctive therapy.These, together with the additional information provided herein, willenable the skilled artisan to determine appropriate administrationprocedures in accordance with embodiments of the invention, withoutundue experimentation.

Routes and Formulations

Compositions comprising EHP cells, including AMP cells, conditionedmedia derived therefrom, cell lysates derived therefrom, or cellproducts derived therefrom, each alone or in combination, or cellsdifferentiated therefrom may be formulated in any conventional mannerusing one or more physiologically acceptable carriers optionallycomprising excipients and auxiliaries. Proper formulation is dependentupon the route of administration chosen. The compositions of theinvention may be packaged with written instructions for use of the cellsin tissue regeneration, or restoring a therapeutically importantmetabolic function. The compositions may also be administered to therecipient in one or more physiologically acceptable carriers. Carriersfor these cells may include but are not limited to solutions ofphosphate buffered saline (PBS) or lactated Ringer's solution containinga mixture of salts in physiologic concentrations.

One of skill in the art may readily determine the appropriateconcentration of EHP cells, including AMP cells, for a particularpurpose. The skilled artisan will recognize that a preferred dose is onewhich produces a therapeutic effect, such as suppressing an inflammatoryor immune response, in a patient in need thereof. Skilled artisans willalso recognize that any and all of the standard methods and modalitiesfor tissue, organ and cell-based transplantation therapies currently inclinical practice and clinical development are suitable for practicingthe methods of the invention. Further, proper doses of EHP cells,including AMP cells, and cell lysates derived therefrom and conditionedmedia derived therefrom (i.e. ACCS), and dosing regimens are easilydetermined by those of skill in the art and need to be empiricallydetermined at time of use based on several variables including but notlimited to disease being treated; patient age, weight, sex, health;other medications and treatments being administered to the patient; andthe like. Routes of administration, formulation, co-administration withother agents (if appropriate) and the like are discussed in detailelsewhere herein. A preferred dose is in the range of about 0.25−2.0×10⁶cells. Other preferred dose ranges are 0.1−10.0×10⁶ cells per squarecentimeter of applied area based on in vitro as well as in vivoexperiments. In a particular preferred embodiment, it has been foundthat relatively small amounts of EHP cells can suppress inflammatory andimmune responses. For example, only 1,000-100,000 AMP cells per reactionin MLRs is sufficient to reduce T-cell response to potent stimulators byover 99% in vitro.

In addition, one of skill in the art may readily determine theappropriate concentration of conditioned media, including, for example,ACCS, for a particular purpose. A preferred dose is in the range ofabout 0.1-to-1000 micrograms per square centimeter of applied area.Other preferred dose ranges are 1.0-to-50.0 micrograms/applied area.

EHP cells, including AMP cells, conditioned media derived therefrom,cell lysates derived therefrom, or cell products derived therefrom, eachalone or in combination, or cells differentiated therefrom can beadministered by injection into a target site of a subject, preferablyvia a delivery device, such as a tube, e.g., catheter. In a preferredembodiment, the tube additionally contains a needle, e.g., a syringe,through which the cells can be introduced into the subject at a desiredlocation. Specific, non-limiting examples of administering cells tosubjects may also include administration by subcutaneous injection,intramuscular injection, or intravenous injection. If administration isintravenous, an injectable liquid suspension of cells can be preparedand administered by a continuous drip or as a bolus.

The compositions of the invention may also be inserted into a deliverydevice, e.g., a syringe, in different forms. For example, the cells canbe suspended in a solution contained in such a delivery device. As usedherein, the term “solution” includes a pharmaceutically acceptablecarrier or diluent in which the cells of the invention remain viable.Pharmaceutically acceptable carriers and diluents include saline,aqueous buffer solutions, solvents and/or dispersion media. The use ofsuch carriers and diluents is well known in the art. The solution ispreferably sterile and fluid to the extent that easy syringabilityexists. Preferably, the solution is stable under the conditions ofmanufacture and storage and preserved against the contaminating actionof microorganisms such as bacteria and fungi through the use of, forexample, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, andthe like. Solutions of the invention can be prepared by incorporatingEHP cells, including AMP cells, conditioned media derived therefrom,cell lysates derived therefrom, or cell products derived therefrom, eachalone or in combination, or differentiated cells as described herein, ina pharmaceutically acceptable carrier or diluent and, as required, otheringredients enumerated above, followed by filter sterilization.

Undifferentiated, partially differentiated or fully differentiated EHPcells, including AMP cells, conditioned media derived therefrom, celllysates derived therefrom, each alone or in combination, may beadministered systemically (for example intravenously) or locally (forexample directly into a myocardial defect under echocardiogram guidanceor by direct application under visualization during surgery). For suchinjections, the compositions may be in an injectable liquid suspensionpreparation or in a biocompatible medium which is injectable in liquidform and becomes semi-solid at the site of damaged tissue. Aconventional intra-cardiac syringe or a controllable endoscopic deliverydevice can be used so long as the needle lumen or bore is of sufficientdiameter (e.g. 30 gauge or larger) that shear forces will not damage anycells being delivered.

Support matrices into which the EHP cells, including AMP cells, and/orcell lysates and/or conditioned media derived therefrom can beincorporated or embedded include matrices which are recipient-compatibleand which degrade into products which are not harmful to the recipient.These matrices provide support and protection for undifferentiated anddifferentiated EHP cells in vivo and are, therefore, the preferred formin which such cells are transplanted into the recipient subjects.

Natural and/or synthetic biodegradable matrices are examples of suchmatrices. Natural biodegradable matrices include plasma clots, e.g.,derived from a mammal, collagen, fibronectin, and laminin matrices.Suitable synthetic material for a cell transplantation matrix must bebiocompatible to preclude migration and immunological complications, andshould be able to support extensive cell growth and differentiated cellfunction. It must also be resorbable, allowing for a completely naturaltissue replacement. The matrix should be configurable into a variety ofshapes and should have sufficient strength to prevent collapse uponimplantation. Recent studies indicate that the biodegradable polyesterpolymers made of polyglycolic acid fulfill all of these criteria(Vacanti, et al. J. Ped. Surg. 23:3-9 (1988); Cima, et al. Biotechnol.Bioeng. 38:145 (1991); Vacanti, et al. Plast. Reconstr. Surg. 88:753-9(1991)). Other synthetic biodegradable support matrices includesynthetic polymers such as polyanhydrides, polyorthoesters, andpolylactic acid. Further examples of synthetic polymers and methods ofincorporating or embedding cells into these matrices are also known inthe art. See e.g., U.S. Pat. Nos. 4,298,002 and 5,308,701.

Attachment of the cells to the polymer may be enhanced by coating thepolymers with compounds such as basement membrane components, agar,agarose, gelatin, gum arabic, collagens types I, II, III, IV and V,fibronectin, laminin, glycosaminoglycans, mixtures thereof, and othermaterials known to those skilled in the art of cell culture. Allpolymers for use in the matrix must meet the mechanical and biochemicalparameters necessary to provide adequate support for the cells withsubsequent growth and proliferation. The polymers can be characterizedwith respect to mechanical properties such as tensile strength using anInstron tester, for polymer molecular weight by gel permeationchromatography (GPC), glass transition temperature by differentialscanning calorimetry (DSC) and bond structure by infrared (IR)spectroscopy, with respect to toxicology by initial screening testsinvolving Ames assays and in vitro teratogenicity assays, andimplantation studies in animals for immunogenicity, inflammation,release and degradation studies.

One of the advantages of a biodegradable polymeric matrix is thatangiogenic and other bioactive compounds can be incorporated directlyinto the support matrix so that they are slowly released as the supportmatrix degrades in vivo. As the cell-polymer structure is vascularizedand the structure degrades, EHP cells may differentiate according totheir inherent characteristics. Factors, including nutrients, growthfactors, inducers of differentiation or de-differentiation (i.e.,causing differentiated cells to lose characteristics of differentiationand acquire characteristics such as proliferation and more generalfunction), products of secretion, immunomodulators, inhibitors ofinflammation, regression factors, biologically active compounds whichenhance or allow ingrowth of the lymphatic network or nerve fibers,hyaluronic acid, and drugs, which are known to those skilled in the artand commercially available with instructions as to what constitutes aneffective amount, from suppliers such as Collaborative Research, SigmaChemical Co., vascular growth factors such as vascular endothelialgrowth factor (VEGF), epidermal growth factor (EGF), and heparin bindingepidermal growth factor like growth factor (HB-EGF), could beincorporated into the matrix or be provided in conjunction with thematrix. Similarly, polymers containing peptides such as the attachmentpeptide RGD (Arg-Gly-Asp) can be synthesized for use in forming matrices(see e.g. U.S. Pat. Nos. 4,988,621, 4,792,525, 5,965,997, 4,879,237 and4,789,734).

In another example, the undifferentiated, partially differentiated orfully differentiated EHP cells, including AMP cells, may be transplantedin a gel matrix (such as Gelfoam from Upjohn Company) which polymerizesto form a substrate in which the cells can grow. A variety ofencapsulation technologies have been developed (e.g. Lacy et al.,Science 254:1782-84 (1991); Sullivan et al., Science 252:718-712 (1991);WO 91/10470; WO 91/10425; U.S. Pat. No. 5,837,234; U.S. Pat. No.5,011,472; U.S. Pat. No. 4,892,538). During open surgical procedures,involving direct physical access to the damaged tissue and/or organ, allof the described forms of undifferentiated, partially differentiated orfully differentiated EHP cell delivery preparations are availableoptions. These cells can be repeatedly transplanted at intervals until adesired therapeutic effect is achieved.

The invention also provides for the delivery of EHP cells, including AMPcell compositions described herein, in conjunction with any of the abovesupport matrices as well as amnion-derived membranes. Such membranes maybe obtained as a by-product of the process described herein for therecovery of AMP cells, or by other methods, such as are described, forexample, in U.S. Pat. No. 6,326,019 which describes a method for making,storing and using a surgical graft from human amniotic membrane, US2003/0235580 which describes reconstituted and recombinant amnioticmembranes for sustained delivery of therapeutic molecules, proteins ormetabolites, to a site in a host, U.S. 2004/0181240, which describes anamniotic membrane covering for a tissue surface which may preventadhesions, exclude bacteria or inhibit bacterial activity, or to promotehealing or growth of tissue, and U.S. Pat. No. 4,361,552, which pertainsto the preparation of cross-linked amnion membranes and their use inmethods for treating burns and wounds. In accordance with the presentinvention, EHP cells may be grown on such membranes, added to themembrane in either an undifferentiated, partially differentiated orfully differentiated form, or conditioned media or cell lysates may beadded to such membranes. Alternatively, amniotic tissue in which AMPcells have not been stripped away may be used to deliver EHP cells to aparticular site. In all cases, EHP cells used in conjunction withamniotic tissue or other matrices can be used in combination with othertherapeutically useful cells and/or cells expressing biologically activetherapeutics such as those described in below.

EHP cells may be genetically engineered to produce a particulartherapeutic protein. Therapeutic protein includes a wide range ofbiologically active proteins including, but not limited to, growthfactors, enzymes, hormones, cytokines, inhibitors of cytokines, bloodclotting factors, peptide growth and differentiation factors. Particulardifferentiated cells may be engineered with a protein that is normallyexpressed by the particular cell type. For example, pancreatic cells canbe engineered to produce digestive enzymes. Hepatocytes can beengineered to produce the enzyme inhibitor, A1AT, or clotting factors totreat hemophilia. Furthermore, neural cells can be engineered to producechemical transmitters.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing a nucleic acid encoding theprotein of interest linked to appropriate transcriptional/translationalcontrol signals. See, for example, the techniques described in Sambrooket al, 2001, “Molecular Cloning: A Laboratory Manual”; Ausubel, ed.,1994, “Current Protocols in Molecular Biology” Volumes I-III; Celis,ed., 1994.

Suitable methods for transferring vector or plasmids into EHP cells orcells differentiated therefrom include lipid/DNA complexes, such asthose described in U.S. Pat. Nos. 5,578,475; 5,627,175; 5,705,308;5,744,335; 5,976,567; 6,020,202; and 6,051,429. Suitable reagentsinclude lipofectamine, a 3:1 (w/w) liposome formulation of thepoly-cationic lipid2,3-dioleyloxy-N-[2(sperminecarbox-amido)ethyl]-N,N-d-imethyl-1-propanaminiumtrifluoroacetate (DOSPA) (Chemical Abstracts Registry name:N-[2-(2,5-bis[(3-aminopropyl)amino]-1-oxpentyl)amino)ethyl-]-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-1-propanamin-trifluoroacetate),and the neutral lipid dioleoyl phosphatidylethanolamine (DOPE) inmembrane filtered water. Exemplary is the formulation Lipofectamine2000™ (available from Gibco/Life Technologies #11668019). Other reagentsinclude: FuGENE™ 6 Transfection Reagent (a blend of lipids innon-liposomal form and other compounds in 80% ethanol, obtainable fromRoche Diagnostics Corp. #1814443); and LipoTAXI™ transfection reagent (alipid formulation from Invitrogen Corp., #204110). Transfection of EHPcells can be performed by electroporation, e.g., as described in Roachand McNeish (Methods in Mol. Biol. 185:1 (2002)). Suitable viral vectorsystems for producing stem cells with stable genetic alterations may bebased on adenoviruses, lentiviruses, retroviruses and other viruses, andmay be prepared using commercially available virus components.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting as the scope of theinvention can only be determined by the claims.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Characterization of Amnion-Derived Multipotent Progenitor(AMP) Cells Used in Studies

For immunological studies, a further characterization of the cellsurface markers of AMP cells, a distinct sub-population of cellsisolated from human amnion that have been cryopreserved, cultured, andpropagated, was performed before examination of their immunologicalcharacteristics. Under these conditions, AMP cells are negative for theCD 117 antigen (c-kit), distinguishing AMP cells from amnioticfluid-derived cells, and are ˜74% positive for CD90 and ˜97% SSEA-4. AMPcells also exhibit staining for CD9 (˜81%), CD10 (˜64%), CD29 (˜100%),CD104 (˜100%), CD49f (˜100%), CD105 (˜12%) and CD44 (˜7%), but arenegative for hematopoietic markers CD34 and CD45 and for the PDGFreceptor CD140b. Further analysis was performed to establish a relevantimmunological profile for AMP cells. AMP cells expressed MHC class 1molecules, however were negative for MHC Class II. AMP cells were alsonegative for the co-stimulatory molecules CD80 and CD86 (B7-1 and B7-2,respectively). The expression of these markers was unaffected byincubation in the presence of IFN-γ. AMP cells were shown to have Fasexpression, possibly providing a mechanism against FasL-expressingactivated effector cells in vivo. Consistent with other findings ofplacenta-derived cells, AMP cells we found to be positive for both PD-L1and PD-L2 after exposure to IFN-γ. Finally, AMP cells express thenon-classical HLA class I antigen HLA-G, a key component of theimmuno-modulatory effects of the amnion itself. HLA-G was present on˜60-90% of cells at the time of isolation, and decreased gradually overpassage. HLA-G expression was up-regulated by the addition of IFN-γ. AMPcells were negative, however, for the non-classical HLA class Ireceptors IL-T2, IL-T3, and IL-T4 at all times tested. These moleculesare up-regulated by HLA-G itself, but the lack of these receptors on AMPcells may indicate the absence of any autocrine regulation by HLA-G onthe cell surface.

Example 2 Immunogenicity of AMP Cells: Mixed Lymphocyte Reaction (MLR).Normal Peripheral Blood Mononuclear Cells vs. HLA-DR (Class II)Mismatched AMP Cells

HLA-G is a non-classical major histocompatability complex (MHC) class Imolecule that has a tissue-restricted distribution. It has been shown toprovide materno-fetal tolerance and is expressed on cytotrophoblasts atthe fetal-maternal interface. HLA-G is also expressed by certain cancerlineages, where it may have a role in providing an escape mechanism tothe immuno-surveillance of the host organism. It has been shown (CMLS,55, 1999, 327-333, incorporated by reference herein) that HLA-G caninhibit MHC-restricted (T-cell) and unrestricted (NK cell) responsesthrough Killing Inhibitory Receptors (KIR) expressed on the T-cell andNK cells. This includes lytic as well as proliferative responses.Therefore, HLA-G has immunomodulatory properties of the cells expressingit and of the environment in which these cells are located.

Applicants have found that AMP cells variably express the HLA-G cellsurface marker upon isolation from the amnion (>60%, n=20); however, theexpression of this marker decreases as the cells are cultured over time.When pro-inflammatory cytokines (IFNγ (100 IU/mL) alone or with TNFα (10ng/mL) and IL1β (10 ng/mL)) are added to the culture, HLA-G expressionis up-regulated. In addition, the MHC Class II markers (i.e. DR, DP, DQ)are not expressed by AMP cells either at isolation or after culture withor without pro-inflammatory cytokines Applicants have also have foundthat AMP cells do not express the co-stimulatory molecules CD80 andCD86, which are pivotal in signaling a normal immune response. Since AMPcells have no MHC Class II or co-stimulatory molecule expression andhave HLA-G expression on the cell surface, it is theorized that AMPcells will not illicit a T helper immune response and may haveimmunomodulatory properties.

To test this hypothesis and determine the immunogenic potential of AMPcells, both at isolation and after culture, the cells were tested in astandard mixed lymphocyte reaction (MLR) (see, for example, Bach, F. H.,Hirschorn, H. “Lymphocyte interaction: A potential histocompatabilitytest in vitro. Science 1964; 143:813-814. Bain, B., Vaz, MR, Lowenstein,L. “The development of large immature mononuclear cells in mixedlymphocyte cultures.” Blood 1964; 23:108-116. Jeras, M. “The role of invitro alloreactive T-cell functional tests in the selection of HLAmatched and mismatched haematopoietic stem cell donors.” TransplantImmunology 2002; 10:205-214, all of which are incorporated herein byreference).

Responder cell populations consisted of ficoll-hypaque-isolatedperipheral blood mononuclear cells. Two normal volunteers (Responder 1and Responder 2) with mismatched HLA-DR loci were chosen. Stimulatorcell populations consisted of AMP cells that were tissue-typed at theDNA level and found to mismatch both normal responders. Normalresponders were also used as stimulators against each other as apositive control. Stimulator cell populations were irradiated with 2000Rads to prevent proliferation during the one-way MLR. Cells were mixedat 100,000 cells responders vs. 100,000 cells stimulators in one well ofa 96-well plate and run in triplicate. Cultures were incubated for 6days and were then pulsed with tritiated Thymidine for 1 day and thenharvested and counted using a beta-scintillation counter. The results ofthis experiment are shown in FIG. 1.

Responder cells are listed as numbers 1-21 on the X axis of FIG. 1.Numbers 1-7 represent AMP cells vs. irradiated (asterisk) stimulatorcells shown in the legend. Numbers 8-14 represent Responder 1 vs.irradiated stimulator cells shown in the legend. Numbers 15-21 representResponder 2 vs. irradiated stimulator cells shown in the legend.

Normal PBMCs did not respond to any of the irradiated stimulator AMPcells shown in FIG. 1. Normal HLA-DR mismatched responders (1 and 2)only had a positive reaction by MLR when cultured with each other. Therewas no significant response of either normal responder vs. irradiatedAMP cells at either the non-passaged time point (P0), or when culturedlonger after one passage (P1). Pro-inflammatory cytokine addition (IFN-γalone or IFN-γ, IL-1, and TNF-α) did not augment a response of eithernormal responders to AMP cells, but these cytokines may have been toxicat the same time. These data suggest that AMP cells are not capable ofgenerating an immune response at the helper T-cell level.

Example 3 Immunomodulatory Effects of AMP Cells: Mitogen Response, MixedLymphocyte Reaction, and Antigen-Specific Memory Response toCytomegalovirus (CMV)

Normal PBMCs respond vigorously to stimulation with the mitogenConcanavelin A (Con A) in a three day culture. To assess theimmunomodulatory effects of AMP cells on Con A stimulation, variousconcentrations of AMP cells were titrated into the assay. Briefly, PBMCswere cultured at 1×10̂5 cells per well in a 96-well round bottom tissueculture plate (BD Falcon, Cat. #08-772-5) in the presence of 1004 Con A(Sigma, Cat. # C5275). The cultures were incubated at 37° C. in ahumidified 5% CO₂ incubator for 2 days, then pulsed overnight with 1 μCitritiated thymidine (³H-dTR, Perkin Elmer, Waltham, Mass.) to measure Tcell proliferation. Cultures were then harvested using a Mach III96-well cell harvester (Tomtec, Hamden, Conn.) and counted using aMicrobeta scintillation counter (Wallac Inc., Gaithersburg, Md.). Theresults were expressed as mean values of triplicate cultures in countsper minute (CPM) and are shown in FIG. 2. AMP cells were co-culturedwith the PBMCs and mitogen at 1:4, 1:16 and 1:64 dilutions. Positivecontrols consisted of PBMC plus mitogen alone. Negative controlsconsisted of PBMC alone and AMP cells alone in culture media. Allcultures were harvested and counted at day 3. Inhibition of T cellproliferation was determined by the percent reduction in CPM from wellscontaining AMP cells vs. the total proliferation of PBMC to Con A.

The one-way MLR was used to assess T cell reactivity vs. allogeneicstimulation. To identify whether AMP cells were immunogenic toallogeneic PBMCs, responder PBMCs were cultured in 200 μl RPMI+5% humanAB serum at a concentration of 1×10̂5 cells per well in a 96-well roundbottom tissue culture plate along with stimulator 2000 rad-irradiatedAMP cells at the same concentration in triplicate. The cultures wereincubated at 37° C. in a humidified 5% CO₂ incubator for 6 days, thenpulsed for 6 hours with 1 μCi tritiated thymidine to measure T cellproliferation. The results were expressed as mean values of triplicatecultures in counts per minute (CPM) and are shown in FIG. 2. Thisexperiment was repeated several times using two normal PBMC populationswith mismatched HLA DR alleles. The positive control included MLRcultures between two HLA-DR mismatched PBMCs. Negative controlsconsisted of responder and stimulator cells alone in RPMI+5% AB serumand responder cells vs. themselves (irradiated). To assess theimmunomodulatory characteristics of AMP cells in vitro, AMP cells wereserially titrated into a MLR between PBMCs isolated from normalvolunteers with mismatched HLA-DR alleles. Stimulator PBMCs and AMPcells were irradiated with 2000 rads to prevent proliferation. AMP cellswere titrated into the MLR at a starting concentration 1:4, and furtherdiluted to 1:16, and 1:64. T cell proliferation was measured asdescribed above. Inhibition of T cell proliferation was determined bythe percent reduction in CPM of wells containing AMP cells vs. the totalproliferation between normal responders and stimulators.

The immunomodulatory effects of AMP cells on the recall memory responseof T cells to Cytomegalovirus (CMV) antigen was also tested. Normal CMVseropositive PBMCs were isolated as described above and cultured in thepresence of soluble CMV antigen at 1×10̂5 cells per well in 96-wellplates in triplicate. 2000 rad-irradiated AMP cells were titrated intothe cell culture wells at a starting concentration of 1:4, and furtherdiluted to 1:16 and 1:64. These cultures were incubated at 37° C. in ahumidified 5% CO₂ incubator for 6 days, then pulsed for 6 hours with 1μCi tritiated thymidine to measure T cell proliferation. The results areexpressed as mean values of triplicate cultures in counts per minute andare shown in FIG. 2. The positive control consisted of PBMCs in thepresence of CMV antigen alone. Negative controls consisted of responderPBMCs and AMP cells alone in culture media Inhibition of T cellproliferation was determined by the percent reduction in CPM of wellscontaining AMP cells vs. the total proliferation of PBMC to soluble CMVantigen.

Results: In the absence of AMP cells, T cells responded vigorously tostimulation with Con A, allogeneic MLR, and to CMV antigen. Significantinhibition occurred when AMP cells were titrated into these assays in adose-dependent manner (p<0.01). This dose effect was most prevalent forin the recall antigen assay, where seropositive T cells were inhibitedby an average of 95% by AMP cells titrated into the assay at a dilutionof 1:4. Results were reproducible when seven CMV seropositive responderswere tested against soluble CMV antigen in the presence of fourdifferent AMP populations. This effect was also observed in theallo-antigen MLR, where average inhibition reached 80.1% inhibition.Again, seven different sets of responders and mismatched stimulatorswere used with four different AMP populations in these assays. Incontrast, Con A response reached only 51.6% inhibition in the presenceof AMP cells at this dilution. Six different responders were testedagainst soluble mitogen with four different AMP cell populations. Theseinhibitory effects of AMP cells were unaffected by the addition orpre-incubation of AMP cells with IFN-γ. IFN-γ also did not up-regulateMHC class II expression on AMP cells at any time, but HLA-G expressionwas up-regulated to over 60% on AMP cells surface (FIG. 2). As AMP cellswere further diluted to 1:16, there was still significant dose-dependentinhibition in all three assays tested, but this effect was not equallydistributed. The highest inhibition was observed with CMV-specificresponses at an average of 62%, whereas the allo-antigen and the mitogenresponses dropped to an average of 47% and 34% inhibition, respectively.At the final dilution of AMP cells used in these assays (1:64), only therecall response to CMV remained inhibited by more than 50%.

Example 4 Effects of Passaging and Dose Response of AMP Cells onAllo-Antigen MLR

AMP cells were isolated from various time points in culture andpassaged. P0 is the earliest time point and are non-passaged cells. P1,P2, and P3 refer to passage numbers with P1 being cells taken after thefirst passage, P2 the second, and P3 the third. After collection, theAMP cells were irradiated and placed into a normal MLR betweenresponders 1 and 2 at 1×10̂5 starting concentration. AMP cells were thenserially diluted from this starting concentration down to 1:64 and addedto the MLR to see the effects of lower cell numbers on inhibition. FIG.3 shows the results of this experiment.

The total uninhibited response between normal responders 1 and 2 by MLRare shown as numbers 1-4 on the X axis of FIG. 3. Inhibitory effects ofP0, P1, P2, and P3 AMP cell titrations are represented by numbers 5-32on the X axis.

The results show significant inhibition of the normal MLR with no impactof passage time of the AMP cells. MLR was inhibited by up to 89% at thehighest AMP cell dilution. A titration effect was observed, with AMPcell inhibition remaining over 20% even at the 1:64 dilution. In theseAMP cells, HLA-G expression was measured at ˜52% at isolation or P0 andwas still detected at P3 being (˜12%).

Example 5 Effects of Passaging and Dose Response of AMP Cells on MemoryResponse to Cytomegalovirus (CMV)

The immunomodulatory characteristics of AMP cells was also assessed by asecond experiment in which the AMP cells were irradiated and seriallydiluted as before, but instead of culturing in a normal MLR, the cellswere titrated into a recall antigen memory response to Cytomegalovirusassay. Responder 2 is also a CMV sero-positive responder. When culturedwith isolated CMV antigen, there is a high response as measured by astimulation index of H³ Thymidine incorporation. This is a classicalmemory response to an antigen that these cell lineages were previouslyexposed to. When AMP cells were titrated into this system, there was anabrogation of this memory response, as shown by FIG. 4.

The total uninhibited response between the sero-positive responder andCMV antigen is represented as numbers 1-4 on the X axis of FIG. 4. Theeffect of titrated AMP cells at P0, P1, P2, and P3 is represented bynumbers 5-32 on the X axis of FIG. 4.

The normal CMV response was inhibited by greater than 90% using thefirst 3 serial dilutions of the AMP cells titrated into the culture(1:1, 1:2, and 1:4). This inhibition steadily dropped with furthertitrations. There was no significant effect of passage number on theinhibition results shown. Taken together, these data illustrate that AMPcells appear to have an immunomodulatory function and can down-regulatethe immune response.

Example 6 Immunomodulatory Effect of the AMP Cells is not a CytotoxicEffect

To confirm that the effect seen in is an immunomodulatory effect of theAMP cells and not a cytotoxic effect, co-culture experiments wereperformed to assess responder growth and viability. The results of thisexperiment demonstrate that PBMC responders co-cultured with AMP cellshave slightly lower viability than PBMC responders alone. PBMCresponders to allogeneic PBMC cultured with AMP cells had a slightdecrease in viability as well. However, PBMC responders to allogeneicPBMC responders without AMP cells also showed some decrease inviability. This decrease in viability may have been due to inappropriatelength of experimental culture.

Example 7 Effect of AMP Cells on Growth Factor-Induced Cell Culture:IL-2 T Cell Blasts and Epstein-Barr Virus (EBV)-Transformed B Cells

AMP cells were analyzed for potential immunomodulation of pre-activatedT and B cell cultures. IL-2-dependent T cell blasts were produced byculturing 10×10⁶ PBMC in the presence of 10 μM Phytohaemagglutinin (PHA,Sigma Cat. # L2646) and 10 U rhIL-2 (Peprotech, Cat. #200-02) over aperiod of 5 days in 20 mls RPMI+5% human AB serum in 25 cm² tissueculture flasks (BD Falcon, Cat. #08-772-18). IL-2-dependent cells werethen collected by centrifugation and washed once and transferred to96-well tissue culture-treated plates at a concentration of 1×10⁵ cellsper well in fresh RPMI+5% human AB serum. Cells were then re-exposed toIL-2 for three days and measured for proliferation by uptake of ³H-dTRas described above. AMP cells were added to these cells at this time ata titration dilution of 1:5, 1:10, 1:50, 1:100, 1:500, and 1:1000 toassess immunomodulatory effects. The positive control consisted of Tcell blasts in culture media in the presence of IL-2. Negative controlsconsisted of T cell blasts in culture media without IL-2 and AMP cellsalone in culture media Inhibition of blast proliferation in response toIL-2 was determined by the percent reduction in CPM of wells containingAMP cells vs. the total proliferation of T cell blasts to IL-2.

Transformed B cell blasts were created by culturing PBMC in the presenceof Epstein-Barr virus as previously described (Pope, J., Scott, W.,Moss, D., Human lymphoid cell transformation by Epstein-Barr virus.National Review of Biology, 1973. 246: p. 140-141). These pre-activatedblasts exhibit self-sustained proliferation when grown in mediacontaining fetal calf serum. Blasts were cultured at 1×10⁵ cell per wellin RPMI+10% fetal calf serum. AMP cells were added to these cells at thesame dilutions as above to assess effects on B cell blast proliferation.The positive control was B cell blasts in media alone. Negative controlsconsisted of B cell blasts alone in media without fetal calf serum andAMP cells alone in culture media. Inhibition of blast proliferation wasdetermined by the percent reduction in CPM of wells containing AMPs vs.the total proliferation of B cell blasts in media with fetal calf serum.

Results: The IL-2 T cell blasts proliferated in response to exogenousIL-2 with a range of 3560-12811 CPM. There was no inhibition ofproliferation of the T cell blasts to re-exposure to IL-2 using anydilution of AMP cells tested. Indeed, the range of proliferativeinhibition was 3472-12041 CPM, respectively (n=3). These results wereconsistently repeated with any dilution of AMP cells used, although thevariability was high due to large differences in T cell blastproliferation. Similar results were found when AMP cells wereco-cultured with B cell blasts in the presence of fetal calf serum inthe culture media (n=6). One example showed B cell proliferation inculture media alone at 1259 CPM. Upon the addition of AMP cells at a 1:4ratio (the highest amount), there was no inhibitory effect with CPMremaining at 1701. At no dilution did AMP cells inhibit B cell blastproliferation to growth factors in culture media. Therefore, there was atotal lack of dose-dependent inhibition by AMP cells seen on growthfactor-activated cells.

Example 8 Transwell Assays and Conditioned Media

Non-irradiated AMP cells were plated into the upper chamber of 24-welltranswell plates (Corning, 4 μm pore size, Cat. #3413) at a confluentdensity of 5×10⁵ AMP cells per well. Mitogen stimulation assays were setup in the lower chambers consisting of 5×10⁵ PBMC isolated from normaldonors and 10 μg/ml Concanavelin A. After 3 days in culture, cells fromthe lower chamber were transferred to 96-well round bottom plates intriplicate (200 μl/well) and pulsed with ³H-dTR and harvested 18 hourslater. Comparison groups consisted of normal PBMC plus mitogen alone,with AMP cells titrated into the wells at 1:5, 1:50, and 1:500 dilution,versus AMP cells at the same titrations separated from the PBMC andmitogen by the transwell membrane apparatus. Data are represented aspercentage of proliferation (proliferation index), where T cells in thepresence of mitogen stimulation alone equaled 100%. Inhibition inproliferation index was calculated by subtracting the percent inhibitionof cultures in the presence of AMP cells plus stimulation from cultureswith stimulation alone.

Conditioned media was removed from AMP cell cultures after growthreached near-confluency in T-75 tissue culture flasks (FisherScientific, Cat. #13-680-58). Conditioned media was immediately added tomitogen, MLR, and recall antigen assays described above by serialdilution yielding final dilutions of 1:2, 1:4, and 1:8 within eachexperiment. Positive controls included PBMC plus mitogen alone, PBMCplus irradiated allo-PBMC alone, and PBMC plus CMV antigen alone.Negative controls were PBMC in culture media alone and PBMC inconditioned media alone at each dilution with culture media. Data arerepresented as percentage of proliferation (proliferation index), whereT cells in the presence of mitogen stimulation alone equaled 100%.Inhibition in proliferation index was calculated by subtracting thepercent inhibition of cultures in the presence of AMPs plus stimulationfrom cultures with stimulation alone.

Results: In the transwell system, AMP cells were unable to suppressPBMCs from proliferating in response to mitogen. Normal PBMC response tomitogen alone represents a proliferation index of 100% (control). Asshown above, AMP cells inhibit this response in a dose-dependent manner.Here, AMP cells were titrated into the assay at dilutions of 1:5, 1:50,and 1:500. AMP cells reduced the stimulation index from 100% down to anaverage of 50±8.9% at 1:5, 56.7±7.4% at 1:50, and 72±10.8% at 1:500.When AMP cells were separated from PBMC and mitogen via transwells,there was no inhibition in the stimulation index. Indeed, at thedilution of 1:5 AMP cells, the proliferation index actually increased by19.1±14.6 above the control. At 1:50, there was a drop in proliferationto 90.1±12.1% and at 1:500 there was a slight increase by 2.3±29.5%. Thereason standard deviations were considerably higher with the transwellsmay be because of the assay conditions employed, resulting in unequalamounts of responding T cells being transferred between the plates, thuscontributing to the wide range of CPMs recorded. Regardless, AMP cellswere still unable to significantly inhibit T cell responses to mitogen.Indeed, counts were actually slightly higher in two of the threedilutions examined (1:5 and 1:500).

The immunomodulatory effect of conditioned media taken from AMP cells inculture on mitogen, allo-antigen (MLR) and recall antigen to CMV assayswas also examined. Conditioned media at the highest concentration (1:2dilution) slightly diminished PBMC response to mitogen and had a greaterinhibition to MLR and recall antigen responses, but did not reachstatistical significance due to high variability. There was noinhibition at all at the 1:4 and 1:8 dilutions tested. Taken together,these data suggest that AMP cells require cell-to-cell contact to elicitan immunomodulatory effect and that this effect is not mediated bysoluble factors.

Throughout the specification various publications have been referred to.It is intended that each publication be incorporated by reference in itsentirety into this specification.

1.-18. (canceled)
 19. A method for down-regulating the autoimmuneresponse a subject's peripheral blood mononuclear cells (PBMCs) exhibitwhen they are exposed to an auto-antigen in the subject, the methodcomprising the step of contacting simultaneously the PBMCs, a purifiedpopulation of CD117-negative Amnion-derived Multipotent Progenitor (AMP)cells, and the auto-antigen such that the AMP cells down-regulate theimmune response exhibited by the PBMCs upon exposure to theauto-antigen.
 20. A method for down-regulating the autoimmune response asubject's peripheral blood mononuclear cells (PBMCs) exhibit when theyare exposed to an auto-antigen in the subject, the method comprising thestep of contacting the PBMCs with a purified population ofCD117-negative Amnion-derived Multipotent Progenitor (AMP) cells,subsequent to the exposure of the PBMCs to the auto-antigen such thatthe AMP cells down-regulate the autoimmune response exhibited by thePBMCs to the auto-antigen.
 21. The method of claim 19 or 20 wherein thePBMCs are further contacted with one or more active agents.
 22. Themethod of claim 21 wherein the one or more active agents is selectedfrom the group consisting of a corticosteroid, a cyclosporine, atacrolimus, a sirolimus, a methotrexate, an azathiopine, amercatopurine, a cytotoxic antibiotic, a polyclonal antibody, amonoclonal antibody, an interferon, an opioid, a TNF binding protein, amycophenolate, a FTY720, and another cell type.
 23. The method of claim22 wherein the monoclonal antibody is selected from the group consistingof an anti-T-cell receptor (CD23) and an anti-IL2 receptor (CD25)antibody.